杨树NL-80106转Bt基因植株的获得及抗虫性

通过根癌农杆菌(Agrobacterium tumefaciens)介导将苏云金杆菌(Bacillus thuringiensis)毒蛋白基因Bt转入杨树NL-80106(美洲黑杨×小叶杨,Populus deltoides×Populus simonii),获得了再生植株.PCR及PCR-Southernblotting的分析结果表明,Bt基因已整合到基因组中.部分转基因植株的杀虫实验表明,转基因植株B45和B64对一龄舞毒蛾(Lymantria dispar Linn.)幼虫有明显抗性,饲喂转基因杨树叶片的幼虫死亡率显著高于未转基因的对照植株.     

表达昆虫特异性神经毒素AaIT基因的转基因烟草的抗虫性

经改造的昆虫特异性神经毒素AaIT基因插入植物高效表达载体得到重组质粒pNGY-2。重组质粒中AaIT基因5＇端与烟草花叶病毒(TMV)的Ω序列3＇端融合,受两个串联的35S启动子控制,通过土壤农杆菌LBA4404介导转化烟草NC89,经NPTⅡ选择后再经GUS染色挑选出阳性再生植株,Southern blotting进一步证实了AaIT基因已经整合到烟草基因组中,对棉铃虫(Heliothis armigera)的抗虫实验表明,转基因烟草有显著的抗虫活性。     

玉米丛生芽体系的建立及抗除草剂转基因植株再生

以玉米优良自交系为材料, 利用芽尖分生组织诱导胚状体和丛生芽, 建立起一种快速有效且取材不受季节限制的玉米丛生芽诱导体系. 以丛生芽组织块为受体, 用基因枪将从拟南芥(Arabidopsis thaliana)突变体中克隆的抗除草剂基因als (acetolac-tate synthase)导入玉米细胞, 经除草剂chlorsulfuron筛选得到抗性组织块并再生植株. PCR检测和Southern杂交表明, 部分再生植株转入了als 基因. 除草剂喷施试验表明, 转基因植株及其子一代具有良好的抗除草剂特性. 建立了一种新的受基因型限制小的玉米离体培养及转基因受体系统, 能快速有效地获得大批转基因植株.     

利用四价抗病基因提高超级杂交稻的抗性

以籼型超级杂交稻(Oryza sativa L. subsp. indica)恢复系9311的胚性愈伤组织为受体, 采用改良的基因枪轰击法将构建在同一植物表达载体上的四价抗真菌病基因(RCH10, RAC22, β-Glu和B-RIP)导入到9311的基因组中. Southern印迹杂交结果表明, 潮霉素抗性再生植株中, hpt标记基因和四价抗真菌病基因是连锁在一起并以孟德尔遗传方式进行传递的. 部分转基因R₁代和R₂代植株分别在烟溪和三亚的典型稻瘟病鉴定圃中表现出对稻瘟病菌(Magnaporthe grisea (Hebert) Barr.)的高抗性. 在高抗的转基因植株中, 多个外源基因均能正常表达. 将高抗稻瘟病的9311 R2代转基因纯系与培矮64S原种进行杂交, 杂种F₁代除对稻瘟病仍可表现出高抗性外, 还同时表现出对稻曲病和黑粉病明显提高的抗性.     

转双抗虫基因烟草的研究

用改造的雪花莲凝集素基因GNAmm与合成的苏云金芽孢杆菌(Bt)毒蛋白cry1Ac基因构建了带有双价基因的植物表达载体,在该表达载体中这两个基因的转录分别受笋瓜PP2启动子(SPP2P)和CaMV 35S启动子的调控。通过根癌土壤杆菌介导转化法,获得了一批抗卡那霉素的转化再生烟草植株。PCR检测及基因组DNA Southern blot\,Slot blot杂交分析的结果表明Gna基因和Bt基因已整合到烟草总DNA中。用Bt毒蛋白抗血清进行Western blot分析,转基因植株均有Bt杀虫蛋白的不同程度的表达。对转化再生烟草的虫试结果表明,在所受试的19株烟草中60%的植株上的棉铃虫在5天内死亡率达到100%,而且存活幼虫的生长发育受到明显抑制;蚜虫抑制生长试验表明,多数转化再生植株具有较强的抗蚜活性,平均能够抑制桃蚜50%～60%的蚜口密度,有的高达80%以上。以上结果表明利用这两个改造过的抗虫基因可以获得既抗虫又耐蚜的转双抗虫转基因植物。     

导入β-1,3-葡聚糖酶及几丁质酶基因的转基因可育油菜及其抗菌核病的研究

利用农杆菌转化法,将组成型表达β-1,3葡聚糖酶及几丁质酶基因的双价植物表达载体pBLGC转化优质甘蓝型油菜品种H165,并得到了抗卡那霉素(Kanamycin,Kan)的再生植株。我们对所得到的抗Kan的再生苗进行了初步分子生物学检测,结果表明,在K15(Kan15mg/L)培养基上的绿苗中有30%为PCR阳性植株,而在K25(Kan10mg/L)培养基上的绿苗有53%的阳性率。对部分PCR阳性的转基因植株进行了点杂交分析,结果均表现出较强的杂交信号,这初步说明外源基因已整合到油菜基因组中。转基因植株的活体接种油菜菌核病菌(Sclerotiniasclerotiorium)试验表明,部分转基因植株比对照显示较强抗病性。     

抗虫转基因欧洲黑杨的培育*

用带有35 S-Ω-Bt-NOS嵌合基因的双元载体的农杆菌LBA 4404转化欧洲黑杨的叶片外植体,共获得54株转化再生植株。用这些再生植株对杨尺蠖(Apcchimia cinerarius)进行毒力测定,昆虫校正死亡率在80一96％的再生植株占总测定植株的15％。部分再生植株对舞毒娥进行测定,表明在5～9天内校正死亡率高达100％,存活昆虫的生长和发育也明显地受到抑制。根据苗木在苗圃中高生长和昆虫校正死亡率采用重心聚类法进行分析,初步选出高生长良好和杀虫率居中的3株再生植株。以选出的植株为主进行了PCR及PCR产物的South—ern blot和再生植株DNA Southern blot测定,结果证明Bt基因已插入到这些植株的DNA上,并表达出苏云金杆菌杀虫蛋白的杀虫活性。     

转修饰cry1Ac基因籼稻明恢81经花药培养获得抗虫DH系

对基因枪法获得的明恢81转修饰的cry1Ac基因当代植株进行花药培养,共接种花药2600枚,获得83份花培植株,其中双倍体植株43份,单倍体植株40份。 PCR结果表明含有cry1Ac基因的植株55份,花培植株群体中转基因与非转基因植株的比值为2∶1（55/28）。进一步结合Southern blot和ELISA分析,于花培植株当代筛选到转基因纯合株系36份。外源蛋白表达量上,花药来源于同一克隆的DH系的不同植株之间基本一致,最高的Cry1Ac含量达0.25%。田间抗虫性试验表明,经花药培养纯合获得的部分转基因纯合系植株对二化螟(Chilo suppressalis)表现出高抗,而且主要农艺性状保持不变。以上结果表明水稻花药培养可以加速转基因的纯合与育种利用。     

果聚糖蔗糖转移酶基因的克隆及耐盐转基因烟草的培育

用PCR方法克隆了枯草杆菌(Bacillus subtilis)果聚糖蔗糖转移酶基因(SacB),将其与克隆自酵母(Saccharomyces cerevisiae)的羧肽酶A的液泡引导信号序列连接得到嵌合基因。测序验证后．插入含NPTⅡ基因的植物双元表达载体pBin438中,经农杆菌介导转化烟草。部分经卡那霉索筛选的抗性芽能在含1％NaCl的Ms培养基上正常生根,而未转化芽不能生根或根生长缓慢。转基因小苗移入盛蛭石的花盆并浇灌含1％NaCl的hoagland＇s营养液,17d后,其中一些转基因烟草植株生长良好,而未转化苗出现明显萎蔫。PR扩增及Nonhem分析证实SacB基因已导入转基因植株并得到转录。此结果表明SacB基因的植物基因工程可提高烟草植株的耐盐性。     

无选择标记和载体骨干序列的Xa21转基因水稻的获得

利用双右边界T-DNA载体通过根癌农杆菌介导法将水稻白叶枯病广谱抗性基因Xa21导入杂交稻重要恢复系C418中。T₀代共获得27个独立转基因株系,通过田间抗性鉴定与PCR分析,有17个株系的Xa21基因分子鉴定为阳性,且对白叶枯病原菌P6生理小种具有抗性。通过对17个株系的后代植株进行田间抗性鉴定,分子标记辅助选择及Southern杂交分析,结果显示4个株系的T₁代植株中能分离出无潮霉素标记基因的Xa21转基因植株。无选择标记Xa21转基因株系的获得率为15%。PCR检测还表明,这些无选择标记的Xa21转基因植株不带有载体骨架序列。通过对转基因后代进一步的抗性鉴定与PCR辅助选择,获得了无选择标记和载体骨架序列的转基因Xa21纯合的抗白叶枯病水稻。     

Regeneration of Populus nigra transgenic plants expressing a Kunitz proteinase inhibitor (KTi3) gene

Transgenic poplar (Populus nigra, cv. Jean Pourtet) plants were recovered as a result of Agrobacterium tumefaciens-mediated transformation performed with EHA105 pBI-KUN strain. Plasmid pBI-KUN contains a 650 bp insert derived from the soybean (Glycine max L.) KTi₃, gene, coding for a Kunitz trypsin proteinase inhibitor. A total of 58 independent transgenic lines were obtained from 200 co-cultivated leaf explants. Southern blot hybridization analysis demonstrated the presence of KTi₃ gene in the poplar genome. Northern blot analysis of different kanamycin-resistant plantlets confirmed the accumulation of KTi₃ mRNA and revealed different levels of expression. The trypsin inhibitory activity was determined in poplar transgenic tissues by means of specific assay. Moreover, the trypsin-like digestive proteinases of the polyphagous moth Lymantria dispar (Lepidoptera, Lymantriidae) and Clostera anastomosis (Lepidoptera, Notodontidae) were detected and inhibited in vitro by Kunitz proteinase inhibitor from selected transgenic plants. Two insect bioassays were performed on P. nigra transgenic plant lines, using larvae of the above mentioned insects. In both cases larval mortality and growth as well as pupal weight were not significantly affected when the insects were fed on transgenic leaves and control leaves, respectively.     

Cd胁迫对舞毒蛾幼虫食物利用的影响及其Cd的外泌机制

为探明植食性昆虫对受重金属胁迫的寄主植物的生理生态响应机制,本研究用Cd胁迫下银中杨的叶片饲养舞毒蛾幼虫,分析舞毒蛾幼虫对食物的利用情况以及其对Cd的排毒代谢机制.结果表明: 取食Cd胁迫下银中杨的叶片后,舞毒蛾3～6龄幼虫体内的Cd浓度和Cd含量均显著高于对照,但随着幼虫龄期增长,其体内Cd浓度显著降低,而Cd含量有不同程度的提高;舞毒蛾幼虫粪便和虫蜕中的Cd浓度均显著高于对照;舞毒蛾3～5龄幼虫的食物消耗率显著高于对照,而转化率显著低于对照;3～4龄幼虫的食物利用率均与对照差异不显著,但在5龄时显著低于对照.说明在Cd胁迫下,舞毒蛾幼虫能通过有效的排毒代谢途径将体内富集的部分Cd排出体外,且高龄幼虫的排毒代谢能力强于低龄幼虫;舞毒蛾幼虫体质量的增加会对体内的Cd浓度形成一种稀释效应;舞毒蛾幼虫能通过调整食物消耗率和转化率之间的比例,来维持其正常生长发育所需的食物利用率,但超过一定限度后仍会造成食物利用率降低.     

Influence of dietary aconitine and nicotine on the gut microbiota of two lepidopteran herbivores

The gut microbiota plays an important role in pheromone production, pesticide degradation, vitamin synthesis, and pathogen prevention in the host animal. Therefore, similar to gut morphology and digestive enzyme activity, the gut microbiota may also get altered under plant defensive compound-induced stress. To test this hypothesis, Dendrolimus superans larvae were fed either aconitine- or nicotine-treated fresh leaves of Larix gmelinii, and Lymantria dispar larvae were fed either aconitine- or nicotine-treated fresh leaves of Salix matsudana. Subsequently, the larvae were sampled 72hr after diet administration and DNA extracted from larval enteric canals were employed for gut microbial 16S ribosomal RNA gene sequencing (338 F and 806 R primers). The sequence analysis revealed that dietary nicotine and aconitine influenced the dominant bacteria in the larval gut and determined their abundance. Moreover, the effect of either aconitine or nicotine on D. superans and L. dispar larvae had a greater dependence on insect species than on secondary plant metabolites. These findings further our understanding of the interaction between herbivores and host plants and the coevolution of plants and insects.     

外源茉莉酸诱导的青杨叶片保护性酶活性变化及其对舞毒蛾幼虫生长发育的影响

为探讨外源茉莉酸对青杨Populus cathayana的诱导抗性及其对舞毒蛾Lymantria dispar的影响, 室内对青杨扦插苗喷施不同浓度的茉莉酸（对照为0.17%丙酮）, 分别在喷施后1, 5和10 d采集叶片分析其保护性酶活性的变化, 并接舞毒蛾幼虫于青杨苗木上观测其长发育情况。结果表明： 0.1 和0.001 mmol/L两种浓度的茉莉酸（JA）处理均使青杨叶过氧化物酶（POD）、 多酚氧化酶（PPO）、 苯丙氨酸解氨酶（PAL）、 胰凝乳蛋白酶抑制剂（CI）和胰蛋白酶抑制剂（TI）活性较对照增加(P<0.05)。取食茉莉酸诱导的青杨苗木后, 舞毒蛾幼虫的发育历期延长, 体重降低。0.1 mmol/L茉莉酸诱导的青杨苗木5 d后接虫, 使舞毒蛾幼虫的发育历期显著延长, 较对照长8 d; 接虫21 d后称重时, 取食茉莉酸诱导的青杨叶片的幼虫体重较对照组降低了50%~100%, 该结果说明外源茉莉酸诱导青杨产生了对舞毒蛾明显的抗虫性。     

向小黑杨转化蜘蛛杀虫毒素基因（英文）

近年来,危害小黑杨Populus simonii×Ｐ.nigra的害虫发生严重,给林业生产造成很大损失。为了提高小黑杨的抗虫能力,避免使用杀虫剂带来的污染,用农杆菌介导法将澳大利亚漏斗蛛Atrax robustus的毒蛋白基因和苏云金芽孢杆菌CryⅠA（b）基因C末端的融合基因BGT转化入小黑杨。PCR 和Southern印记分析转基因植株,结果表明,BGT杀虫基因已经整合在小黑杨基因组上。活性实验表明,取食转基因杨树6天和9天后,舞毒蛾Lymantria dispar 2龄幼虫的校正死亡率分别是37.0%和92.6%。方差分析表明取食转基因和对照杨树的舞毒蛾幼虫体重差异显著。这些结果显示转基因杨树上的舞毒蛾的发育速率受到影响。     

Trabala Vishnou Gigantina Yang (Lepidoptera: Lasiocampidae) Larval Fitness on six Sympatric Plant Species in Sea-Buckthorn Forest

Trabala vishnou gigantina Yang (Lepidoptera: Lasiocampidae) is a major pest that damages the sea-buckthorn, Hippophae rhamnoides. We observed and compared the feeding preferences of T. vishnou gigantina larvae on six sympatric plant species in a two-choice test. We also compared T. vishnou gigantina fitness, as measured by the following variables: larvae weight, developmental period, pupae versus adult weight, longevity, and fecundity rates. Between host and non-host plants, larvae showed a strong preference for their natural host (sea-buckthorn), followed by apricot, poplar, and willow. Caragana and locust were the least preferred plants when the natural host plant was not present. Larvae reared on sea-buckthorn possessed greater pre-pupal weight, had lower mortality, and developed more quickly into heavier pupae than either poplar-raised or willow-raised larvae. Fecundity was highest on sea-buckthorn, second highest on apricot, and lowest on poplar. Longevity (of both females and males) was not significantly different across plant species. These results clearly demonstrate that T. vishnou gigantina larvae are able to distinguish between host versus non-host plants, and that their preference translates to increased fitness. Possible, non-mutually exclusive explanations for observed preference and fitness differences include variation in required nutritional content across plant species or the presence of plant traits (morphological features or chemical metabolites) that negatively affect larval development. While the exact mechanisms are unknown, these data may be useful for the development of appropriate counter-measures to the damage caused by T. vishnou gigantina on sea-buckthorn.     

Within-plant signalling via volatiles overcomes vascular constraints on systemic signalling and primes responses against herbivores

Plant volatiles play important roles in signalling between plants and insects, but their role in communication among plants remains controversial. Previous research on plant–plant communication has focused on interactions between neighbouring plants, largely overlooking the possibility that volatiles function as signals within plants. Here, we show that volatiles released by herbivore-wounded leaves of hybrid poplar ( Populus deltoides  ×  nigra ) prime defences in adjacent leaves with little or no vascular connection to the wounded leaves. Undamaged leaves exposed to volatiles from wounded leaves on the same stem had elevated defensive responses to feeding by gypsy moth larvae ( Lymantria dispar L.) compared with leaves that did not receive volatiles. Volatile signals may facilitate systemic responses to localized herbivory even when the transmission of internal signals is constrained by vascular connectivity. Self-signalling via volatiles is consistent with the short distances over which plant response to airborne cues has been observed to occur and has apparent benefits for emitting plants, suggesting that within-plant signalling may have equal or greater ecological significance than signalling between plants.     

转双抗虫基因三倍体毛白杨外源基因表达及性状相关性分析

用部分改造的BtCry1Ac基因与慈菇蛋白酶抑制剂（API）基因构建的双抗虫基因表达载体,通过农杆菌介导法转化了三倍体毛白杨Populus tomentosa Carr.,获得一批转双抗虫基因株系。对转基因株系的抗虫毒蛋白表达进行了ELISA和Western Blot检测,同时用转基因株系叶片对杨扇舟蛾Clostera anachoreta Fabricius和舞毒蛾Lymantria dispar L.幼虫进行室内饲虫试验,并对各项外源基因表达指标进行了相关分析。结果表明：在检测的28个转基因株系中,对杨扇舟蛾高抗株系占总参试系号的41％,中抗系号占35.0％,低抗系号占24％,对舞毒蛾高抗株系占参试系号的70％。转基因植株可明显抑制存活幼虫的生长发育,且不同转基因株系饲养的幼虫发育存在显著差异。连续两年相关分析表明,不同转基因株系幼虫死亡率间存在极显著相关。转基因植株对舞毒蛾和杨扇舟蛾均表达出抗虫性,并存在极显著相关。ELISA检测结果表明,不同转基因株系Bt毒蛋白表达量存在差异,变化在0.0011％～0.0161％。转基因植株对害虫的杀虫效果与Bt杀虫蛋白的表达量存在显著相关,表明Bt毒蛋白在抗虫效果中占有重要地位。转基因植株对卡那霉素表现出一定的抗性,但与抗虫程度相关不明显,单纯用卡那霉素作为筛选手段并不能完全反映植株的真实抗虫效果。